Richard Haworth

Nonproliferative and proliferative lesions of the rat and mouse female reproductive system.

The INHAND (International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice) Project (www.toxpath.org/inhand.asp) is a joint initiative of the Societies of Toxicological Pathology from Europe (ESTP), Great Britain (BSTP), Japan (JSTP) and North America (STP) to develop an internationally accepted nomenclature for proliferative and nonproliferative lesions in laboratory animals. The purpose of this publication is to provide a standardized nomenclature for classifying microscopic lesions observed in the female reproductive tract of laboratory rats and mice, with color photomicrographs illustrating examples of some lesions. The standardized nomenclature presented in this document is also available electronically on the internet (http://www.goreni.org/). Sources of material included histopathology databases from government, academia, and industrial laboratories throughout the world. Content includes spontaneous and aging lesions as well as lesions induced by exposure to test materials. There is also a section on normal cyclical changes observed in the ovary, uterus, cervix and vagina to compare normal physiological changes with pathological lesions. A widely accepted and utilized international harmonization of nomenclature for female reproductive tract lesions in laboratory animals will decrease confusion among regulatory and scientific research organizations in different countries and provide a common language to increase and enrich international exchanges of information among toxicologists and pathologists.

Species-specific Inflammatory Responses as a Primary Component for the Development of Glomerular Lesions in Mice and Monkeys Following Chronic Administration of a Second-generation Antisense Oligonucleotide

Chronic administration of drisapersen, a 2′-OMe phosphorothioate antisense oligonucleotide (AON) to mice and monkeys resulted in renal tubular accumulation, with secondary tubular degeneration. Glomerulopathy occurred in both species with species-specific characteristics. Glomerular lesions in mice were characterized by progressive hyaline matrix accumulation, accompanied by the presence of renal amyloid and with subsequent papillary necrosis. Early changes involved glomerular endothelial hypertrophy and degeneration, but the chronic glomerular amyloid and hyaline alterations in mice appeared to be species specific. An immune-mediated mechanism for the glomerular lesions in mice was supported by early inflammatory changes including increased expression of inflammatory cytokines and other immunomodulatory genes within the renal cortex, increased stimulation of CD68 protein, and systemic elevation of monocyte chemotactic protein 1. In contrast, kidneys from monkeys given drisapersen chronically showed less severe glomerular changes characterized by increased mesangial and inflammatory cells, endothelial cell hypertrophy, and subepithelial and membranous electron-dense deposits, with ultrastructural and immunohistochemical characteristics of complement and complement-related fragments. Lesions in monkeys resembled typical features of C3 glomerulopathy, a condition described in man and experimental animals to be linked to dysregulation of the alternative complement pathway. Thus, inflammatory/immune mechanisms appear critical to glomerular injury with species-specific sensitivities for mouse and monkey. The lower observed proinflammatory activity in humans as compared to mice and monkeys may reflect a lower risk of glomerular injury in patients receiving AON therapy.

Differential Expression of COX-1 and COX-2 in the Gastrointestinal Tract of the Rat

The aim of this study was to use immunohistochemistry with morphometry to investigate COX-1 and COX-2 expression in the normal rat gastrointestinal (GI) tract and examine if sites of ulceration previously observed with long-term COX-2 inhibitor administration in mice correlate with differential COX-1/COX-2 expression. COX-2 positive cells were observed predominantly in the apical lamina propria of intestinal villi with fewer cells in the mucosal epithelium. The highest level of COX-2 expression was observed at the ileocaecal junction (ICJ). COX-2 expression was also present in parasympathetic ganglia of the submucosa and muscularis. In the stomach, the highest grade of COX-2 expression was observed in the apical lamina propria of the fundus adjacent to the junctional ridge. In contrast, COX-1 positive cells within the lamina propria were evenly distributed along the GI tract but were present in higher numbers than COX-2 positive cells. The mean level of COX-1 expression at the ICJ was not significantly different from the ileum and caecum. Evidence that the highest level of COX-2 expression in normal rats is located on the ileal side of the ICJ provides the first mechanism to explain spontaneous ulceration and perforation of the distal ileum in COX-2−/− animals.

The assessment of local tolerance, acute toxicity, and DNA biodistribution following particle-mediated delivery of a DNA vaccine to minipigs.

Particle-mediated DNA delivery was used to administer a DNA vaccine against Hepatitis B to minipigs. The study represented one arm of the safety evaluation program for this product and was designed to assess local tolerance, acute toxicity, and biodistribution of the DNA plasmid. The vaccine was given to 4 groups of minipigs that were sacrificed at 2, 28, 56, or 141 days after treatment. The procedure was well tolerated with mild local skin reactions at 2 days postdosing and no evidence of systemic toxicity. By 28 days the skin lesions had regressed apart from a low grade perivascular mononuclear cell infiltrate in the upper dermis, together with a small number of phagocytosed gold particles. This infiltrate persisted up to 141 days. The expressed HBsAg was detected by immunohistochemistry in keratinocytes (usually in association with an intranuclear gold particle) at 2 days but not at later time points. Polymerase chain reaction (PCR) was used to assay treatment sites and selected internal organs to evaluate biodistribution and persistence of the DNA plasmid. At 2 days the plasmid was detected in the treatment sites and also in the inguinal lymph nodes. At day 57 it was present in the treatment sites only and by day 141 appeared to have cleared. The results from this study demonstrate that particle-mediated gene delivery was well tolerated in the minipig. The biodistribution and persistence of the plasmid was within acceptabl e limits for this type of vaccine. As the minipig is regarded as a good model for humans these data support the concept that particle-mediated DNA delivery will be safe in human clinical applications.

Pulmonary Neuroendocrine Cell Hyperplasia: Identification, Diagnostic Criteria and Incidence in Untreated Ageing Rats of Different Strains

Pulmonary Neuroendocrine Cells (PNEC) are found as clusters called neuroepithelial bodies (NEB) or as single cells scattered in the respiratory epithelium. Pulmonary neuroendocrine cell hyperplasia is recorded in humans and experimentally manipulated rodents. The objectives of this work were to identify the optimal immunohistochemical markers for PNEC in the rat for use on paraffin-embedded, formalin-fixed material and to provide the first comparative incidence of PNEC hyperplasia in untreated 2-year-old rats of different strains. Calcitonin-gene related peptide (CGRP) and protein G product 9.5 (PGP9.5) antibodies identified PNEC consistently and selectively. In contrast, PNEC did not express chromogranin-A or S-100. PNEC hyperplasia was defined as foci of PNEC with greater than 40 nuclei, excluding overlying respiratory epithelium and submucosal PNEC. PNEC hyperplasia was observed at low incidence (0–7%) in untreated 2-year-old Sprague-Dawley, Han Wistar and Wistar rats but not Fischer 344 rats. This is the first report of spontaneous PNEC hyperplasia in rats. The cause of this hyperplasia is unknown, but experimental models that induce PNEC hyperplasia by causing bronchiolar cell injury are discussed. PNEC neoplasia in the rat is unreported in the literature and was not observed in animals examined in this study.